Non-mammalian vertebrate embryos as models in nanomedicine

Nanomedicine: Nanotechnology, Biology, and Medicinexx (2014) xxx–xxx

nanomedjournal.com

Non-mammalian vertebrate embryos as models in nanomedicineMartina Giannaccini, PhDa,b,⁎, Alfred Cuschieri, MDb, Luciana Dente, MSca, Vittoria Raffa, PhDa,b

aDepartment of Biology, Cell and Developmental Biology Unit, Università di Pisa, Pisa, ItalybInstitute of Life Science, Scuola Superiore Sant'Anna, Pisa, Italy

Received 22 June 2013; accepted 23 September 2013

Abstract

Various in vivo biological models have been proposed for studying the interactions of nano-materials in biological systems.Unfortunately, the widely used small mammalian animal models (rodents) are costly and labor intensive and generate ethical issues andantagonism from the anti-vivisectionist movement. Recently, there has been increasing interest in the scientific community in the interactionsbetween nano-materials and non-mammalian developmental organisms, which are now being recognized as valid models for the study ofhuman disease. This review examines and discusses the biomedical applications and the interaction of nano-materials with embryonicsystems, focusing on non-mammalian vertebrate models, such as chicken, zebrafish and Xenopus.© 2013 Elsevier Inc. All rights reserved.

Key words: Nano-materials; Zebrafish; Xenopus; Chicken; Embryos

By definition, nano-materials (NMs) exhibit dimensions at orbelow 100 nm.1 At this scale, materials gain specific propertieswith respect to their bulk equivalents. Thus, NMs have arelatively larger surface area when compared to bulk material,and for this reason, they are more chemically reactive. Indeed,some materials are inert and become reactive only in their nano-scale form. In addition, the nano-scale has a marked effect on thestrength and electrical properties because quantum effectsdominate the behavior of the materials with respect to theiroptical, electrical and magnetic properties. For instance, carbonin nanotube form demonstrates magnetic properties,2 and gold(Au) and silver (Ag)3,4 nanoparticles (NPs) that exhibit tailoredproperties emit fluorescence. These striking characteristicscombined with the ability to interact with biomolecules of asimilar size have aroused interest in their potential for biomedicaland clinical applications.5,6 Various in vitro and in vivobiological models have been proposed for studying the in-teractions of nano-materials in biological systems. In vitrostudies, which are based on cell culture, are rapid, efficient, andlow cost. However, results from these studies provide an

This work was partially funded by Fondazione Pisa as part of MBIADproject and by the Italian MoH/Regione Toscana as part of SILS project.

⁎Corresponding author: Martina Giannaccini, Department of Biology,Cell and Developmental Biology Unit, Università di Pisa, Pisa, Italy.

E-mail address: [email protected] (M. Giannaccini).

1549-9634/$ – see front matter © 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.nano.2013.09.010

Please cite this article as: Giannaccini M., et al., Non-mammalian vertebrate ehttp://dx.doi.org/10.1016/j.nano.2013.09.010

incomplete assessment of the interactions with the wholeorganism. Studies using in vivo systems carry a greaterreliability, address the overall effect on the physiology andanatomy of the organism, and thus constitute a more immediatelyrelevant platform for translational clinical studies. Although inwidespread use, small animal models (rodents) are costly andlabor intensive and have generated resistance in life scienceresearch from the anti-vivisectionist lobby. Such issues andconcerns can be allayed using non-mammalian embryos for invivo studies involving nano-materials. Developmental biologyoffers powerful models for the study of functional interactionsbecause embryos are particularly sensitive indicators of adversebiological events. Moreover, embryos provide a usefulplatform to define the mechanism of action of any deleteriouseffects, which result from exposure to NMs.7,8 Because highlycoordinated cell-to-cell communications and molecularsignaling are required for normal development, any perturbationsby nano-materials will disrupt orderly embryogenesis, resultingin abnormal development that is manifested as morphologicalmalformations, behavioral changes and may even causeembryonic death. In addition, it is known that manygenes involved in developmental pathways are de-regulated inmany human cancers and are thus useful subjects to assesstherapeutic targets.9-11

This review addresses the use of embryonic systems tostudy the in vivo interactions with nano-materials, focusingon non-mammalian vertebrate embryo (NMVE) models that

mbryos as models in nanomedicine. Nanomedicine: NBM 2014;xx:1-17,

mailto:[email protected]

http://dx.doi.org/10.1016/j.nano.2013.09.010





Figure 1. (A) Different developmental stages and adult stage of zebrafish (not to scale). One-cell embryo: 0.7 mm; female and male adults: 4-6 cm. (B)Different developmental stages and adult stage of X. laevis (not to scale). One-cell embryo: 1-1.2 mm; female adult: 13 cm; male adult: 8 cm. (C) Differentdevelopmental stages and adult stage of chicken (not to scale). One-cell embryo: 2-3 mm; female adult: 42-46 cm; male adult: 65-75 cm.

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are most commonly used, namely the zebrafish (Daniorerio), frog Xenopus laevis and chicken (Gallus gallus)(Figure 1).

NMVE models

Zebrafish

The zebrafish has been increasingly used as a vertebratemodel for assessing the toxicity of drugs and chemicals.Specifically, the zebrafish model has been shown to be effectivein predicting adverse drug effects, with a good correlationbetween data derived from zebrafish analyses and data availablefrom either human clinical trials or animal pre-clinical testing.12

Thus, the zebrafish has become a useful model for rapidscreening of the teratogenic and toxicological effects of drugsand nano-materials.13-16

The zebrafish produces a large number of embryos with eachfecundation (hundreds), thereby providing the required statisticalpower for analysis, in addition to facilitating the collection ofmaterial for studies. Moreover, zebrafish exhibits a very rapidembryonic development with the first stages of developmentbeing completed within the first 24 hours post-fertilization (hpf)(pharyngula). The embryo fully develops at 120 hpf (larva) andthe entire generation time is approximately three-four months,which is similar to that of rodents (10 weeks). The costsassociated with zebrafish as a model system are significantlylower because zebrafish is much smaller (0.7 μm zygote to4 mm) than mammals and requires less expensive husbandry.Compared to mammalian-based studies, zebrafish experiments

require much less material to assess the nanomaterial–biologicalinteractions and for toxicity studies. Moreover, the highfecundity and short developing time of zebrafish reduce thecost and completion times of these experiments, particularly forin vivo high-throughput screening studies. Because zebrafishembryos are transparent during the developmental stage, theyenable a direct non-invasive assessment of the growth of theirinternal organs, morphogenetic tissue movements, cellularinteraction and subcellular dynamics.17-19

Another favorable feature of zebrafish is that its genome isclosely related to that of humans. Thus, a remarkable similarityin the molecular signaling processes, cellular structure, anatomy,and physiology has been observed among zebrafish and otherhigh-order vertebrates, including humans.20-22 Because thefundamental processes of vertebrate development are highlyconserved across species, primary developmental mutationsidentified in zebrafish have close counterparts in mammals,including humans.23 Most importantly, the zebrafish researchcommunity has developed a range of resources, including mutantstrains, cDNA clone collections, and complete genome sequenc-ing (www.zfin.org, the zebrafish model organism database).Finally, different transgenic zebrafish lines can be easilygenerated.24-26 Taken together, these features render thezebrafish an effective model for the improved understanding ofhuman diseases.23

X. laevis

X. laevis is one of the principal animal model systems used indevelopmental biology since 1950 and much of our knowledgeregarding the mechanisms of early vertebrate development are

http://www.zfin.org

Figure 2. CAMmodel assay in chicken. (1) Preparation: a window is made in the shell to expose the CAM; (2) treatment, e.g., implantation of cancer cells (A) orinjection of pro- or anti-angiogenic factors (B); (3) outcome: (C) tumor growth, (D) neo-angiogenesis, (E) vessels obliteration and regression.

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derived from these studies. Xenopus shares with zebrafish mostof the favorable features previously described, such as largenumbers of embryos with each fecundation (thousands), a veryshort early development time (3 days to reach tadpole), andexhibits external development and close homology with humangenes. However, the embryos are not transparent and theproduction of transgenic lines is difficult due to the Xenopuspseudotetraploid genome and long generation time, althoughrecently, an increased production of transgenic animals has beenreported.27,28 Despite these drawbacks, this model enables thestudy of gene function, and injection of synthetic mRNAs orantisense oligonucleotides. Furthermore, their larger size (from1-1.2 mm (zygote) to 1 cm (120 hpf)) enables an easiermanipulation and microsurgery compared to zebrafish. Further-more, the Xenopus research community has developed anorganism database centralized resource of genomic, develop-mental data and community information (www.xenbase.org).Since 1983, X. laevis has also been known as an ecotoxicolog-ical model because of the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) test. The three endpoints of this test, i.e.,mortality, malformations and growth inhibition, make it apowerful and flexible bioassay for evaluating pollutants inwater, included NMs.29

Chicken

The chicken embryo holds the longevity record as theexperimental model for studies in developmental biologybecause it dates back to Aristotelian anatomical studies.Currently, the chicken embryo is extensively used to study

angiogenesis, anti-angiogenesis and tumor angiogenesis30 due toits chorio-allantoic membrane (CAM), which is the mainstay forin vivo angiogenesis assays and is a model test system forexperimental blood vessel formation and development31

(Figure 2).CAM is a convenient model for monitoring the modifications

of the vasculature because it is accessible to intravenousadministration of chemical substances and its transparentsuperficial layers enable the performance of studies of structuralchanges in blood vessels.32,33 In addition, transgenic chicks areemployed as bioreactors for the production of recombinantproteins. They exhibit many advantages compared to mammals,such as a short generation time (20 weeks), lower cost, andincreased fecundity34 moreover, the glycosylation patterns ofsome chicken proteins are reported to be more akin to humansthan to other mammals.35 The advantages of these models aresummarized in Table 1.

NMVE as alternative models to contribute to the 3Rs

The 3Rs (Replacement, Refinement, Reduction) are a widelyaccepted ethical framework for conducting scientific experi-ments using animals. In accordance with the Directive 2010/63/EU on the protection of animals used for experimental and otherscientific purposes, animal experiments must be replaced withalternatives whenever possible. The number of animals used inexperiments should be reduced to the minimum required forobtaining valid results. The Directive also stresses that animalsuffering must be avoided or kept to a minimum.36 Thisparticularly applies to animal experiments involving species,

http://www.xenbase.org

Table 1Comparative advantages of different animal model systems in biomedical research.

Human zygote Mouse zygote Zebrafish zygote Xenopus zygote Chicken zygote

Size 0.1 mm 0.1 mm 0.7 mm 1-1.2 mm 2-3 mmExternal development No No Yes Yes YesRapid development No No Yes Yes NoAvailability per fecundation 1-2 5-15 strain dependent Hundreds Thousands 1-2 (per day)High degree of homology tothe human genome

- Yes Yes Yes Yes

Well characterized transcriptomes No No Yes No NoAvailability of transgenic embryos No Yes Yes Yes but more rare YesModel for studying the effectsof exogenous agents on development

No Yes Yes Yes Yes

Small quantity of NMs neededto fully evaluate biological responses

No No Yes Yes No


which are closest to human beings. Scientists have madesubstantial progresses in the implementation of alternativemethods to the use of animals for research. Live animals arebeing replaced by non-animal models, such as cell-based,organotypic or ex vivo or in silico models (mechanical orcomputer models).37-40 A large number of models for tissue-specific toxicological effects or human diseases are beingproposed.41,42 Although these models are reproducible withhigh-throughput and are of undoubted benefit for the significantevaluation of nanomaterial-cell interactions, they do not fullyreproduce the results obtained in vivo because of problems withthe absorption, distribution, metabolism and excretion (ADME)by the organism as a whole because this cannot be predictedusing cell-based models (Table 2). Although the use of animals isunavoidable for these in vivo studies, the replacement of 'higherorder' animals with 'lower' animal species is a potentialsolution.43 The general principle, which has advanced sincethe early 1900s by the philosopher-scientist Albert Schweitzer, isto avoid harming sentient animals whenever possible.44

Different species, which can be used for experimentation havebeen catalogued on a scale of 1 to 5, ranging from apparentlyinsensitive low forms of animal life to highly sensitive andintelligent mammals:

(1) low sensibility/consciousness: mollusks;(2) some sensibility: cephalopods, fishes, amphibians;(3) sentient, but potentially limited in consciousness:

reptiles;(4) sentient and conscious: mammals, birds;(5) sentient, highly intelligent and precognitive: primates,

carnivores, cetaceans.45

In this context, the use of NMVE models does not require alicense (i.e., not regulated by procedures) from fecundation to thetime at which they become capable of independent feeding(rather than being dependent on the food supply from the yolk),which in zebrafish has been established at 5 day post fecondation(d.p.f.), in Xenopus at stage 45 and in chicken at 21 d.p.f.Experimentation during these life stages does not cause anyissues; although the organs are well developed enablingfunctional studies, the central nervous system remains relativelyprimitive and these embryos are considered to not be sufficiently

sentient or experience nociceptive sensations when subjected toexperimental procedures (group 1). Thus, NMVEs can be used atunlicensed stages to generate in vivo data in specific organs as analternative to the use of mammals.

The use of these models has the potential to dramatically reducethe large number of small mammals used in the drug developmentprocess. According to a report by the European Commission inNovember 2007, a total of approximately 12.1 million animalswere used for scientific purposes in 2005 in Europe, of which 79%were mammals.46 NMVEs could thus provide a low-costalternative to bridge the “gap” between in vitro and in vivo studieswith a very significant reduction in the number ofmammals used inlife science and pharmaceutical research, including drug develop-ment and nanomedicine research.

Application in nanotoxicology

The large increase in nanomaterial production for industrial,cosmetic, biomedical and research use has resulted in asignificant risk of exposure of NMs to humans. This underliesthe importance of studies on the toxic potential of NMs to humanhealth. Scientific knowledge on mechanisms of NP cell-interaction has accumulated within the past decade, and hasdemonstrated that cells readily take up NPs. However, thesubsequent intracellular mechanisms and pathways underlyingthis process remain incompletely understood. In addition, thereare many variables that influence the intracellular interactionswith nano-materials, e.g., material, size, shape, surface, charge,coating, dispersion, agglomeration, aggregation, concentration,among others, with a vast reported literature on this topic.47,48

The most extensively used NMVE model in nanotoxicologyis the zebrafish by virtue of the advantages listed in Table 1,7

although more recently, Xenopus has also been exploited forsuch studies.49 NMs can be administered to embryos in threeways: by microinjection, exposure and feeding. Because feedingand exposure are the most common contamination routes inhumans (involuntary exposure), studies in the reported literaturemainly focus on these routes.

In the following account, this review will address some keyissues including: can the results from NMVE models validate

Table 2Comparative properties of different model systems in research.

NM-cell interaction PK, ADME Human disease model Costs Ethical issue

2D in-vitro culture Optimal None None Low None3D in-vitro culture Optimal None Low Low NoneEx-vivo cultures/explants Optimal Low Medium High MediumNon-mammalian embryos Optimal Optimal Medium Medium LowMammals Optimal Optimal High High High

PK: Pharmacokinetics; ADME: absorption, distribution, metabolism and excretion.


and confirm in vitro results at the whole organism level? Haveconsistent equivalent data been obtained from experiments onmammals? Why are NMVEs superior to cell culture models?There is a caveat in addressing these questions because attemptsof a correlation of data between separate independent studies areoften difficult, particularly with studies concerning the sameNMs due to the different metrics used (e.g., mass-based or mass-surface area ratio). Specifically, the flaws in these comparativestudies occur for two main reasons. The first is that NMs aresupplied from various sources: although they are of similarchemistry, size and shape, NMs are often of different purities andphysical structure, which ultimately affect their physical orbiological behavior. Second, investigative protocols and assaysused for NMs are very diverse and lack standardization. Theneed for accepted minimal standards for the analysis ofnanomaterials is well recognized50,51 and although a numberof tools are under development, there remains a lack ofestablished standardized protocols and measurement indices inthe current literature. This most likely accounts for the apparentcontroversy and increases the difficulty in making validcomparative analysis between different reported studies testingthe same or similar NMs.

Despite these limitations and the limited number of reportedstudies using NMVE models for toxicological investigation, theavailable results based on NMVE models are generallyconsistent with those reported from cell culture experimentsand studies using mammals. Thus, reports of studies usingzebrafish have shown that Ag-NPs are more toxic than Au-NPs.52,14 The same finding was reported by in vivo studies inrodents53 as well as by in vitro cell culture studies.54 For metaloxide nanoparticles, TiO2 was reported to be less toxic than CuOand ZnO, based on studies on Escherichia coli,55 yeast56 and inhuman cell lines.57,58 A similar trend was observed in Xenopus49

and in zebrafish59 where CuO-NPs were confirmed to be moretoxic than TiO2 and ZnO. Studies performed on rodents revealedthat size was a key parameter that affected the toxicity inducedby NMs, most likely due to the increased reactivity of thematerials, as exemplified in a study performed in mice, whichshowed that the toxicity of silica-NPs increased with a decreasein particle size.60 Similar observations have been reported inzebrafish treated with Au-NPs, chitosan-NPs and Ni-NPs.13,61,62

The shape of NMs is another crucial parameter that affects thebiological behavior and activity of numerous NMs with goldnanostructures63,64 and carbon nanostructures,65 which are themost commonly investigated in studies involving mammaliancell culture66 and rodents.67 Similarly, studies performedon zebrafish embryos confirmed that the toxicological response

is affected by NM shape, as exemplified by the dendriticstructure of Ni-NMs, which caused significantly more malfor-mation and mortality than spherical Ni-NPs of the same size inzebrafish embryos.62

As expected, the in vitro results correlated well with dataobtained from NMVE models, but the latter provides furtherinsight on the mechanisms used by the whole organism, which invitro cell cultures cannot offer. In a study, which analyzed thetoxicity of 30 reference chemicals in fishes, a comparison of thedata in vitro68 and in vivo69,70 demonstrated a major sensitivityin the whole organism assays.68 Specifically, the EC50 valuesobtained using the cellular tests were 10 to 20 times higher thanthe LC50 values obtained for fishes. Moreover, there were alarge number of specific cases demonstrating that the NMVE invivo studies enhanced the knowledge achieved with the in vitrostudy. This is exemplified by an in vitro study that reported onlya small difference among the toxicological potency of 6 differentantifungal agents in vitro,71 while the difference in the relativepotencies was greater in zebrafish72 and rodents.71 This may bedue to differences in the absorption, distribution, metabolism andelimination (ADME) properties of the different compoundsin vivo.

Another study performed in zebrafish embryos reported thatpositively charged Au-NPs caused high mortality, whereasnegatively charged Au-NPs were associated with a highincidence of malformations. In contrast, neutrally charged Au-NPs did not adversely affect embryonic zebrafishdevelopment.73,74 This finding amplified the results of aprevious study using mammalian cell culture, which hadshown that EC50 for charged Au-NPs was substantially lowercompared to the neutrally charge Au-NPs. This cell culture studyhad demonstrated significant mitochondrial stress followingexposure to the charged Au-NPs, but not to the neutral Au-NPs.Up- or down-regulation of DNA damage related gene expressionsuggested a differential mechanism for cell death, where chargedNPs induced cell death via apoptosis and neutral NPs inducedcell death via necrosis.75 Thus, while the in vitro studyelucidated the molecular mechanisms for cell toxicity, theNMVE study enabled the correlation of the effect of surfacecharge to the different uptake/excretion mechanisms – thus, theelimination processes for the neutral and negative particles weremore efficient than uptake. In contrast, the positively chargedNPs were either not eliminated following uptake or werecontinuously replaced at the same or at a faster pace than theirelimination rate.73

NMVE models represent a more sensitive tool for theassessment of toxicity. The results achieved using cell culture

Table 3Toxicity key factors valuation in non-mammalian animal model systems.

C60-NPs Ag-NPs Ni-NPs CuO-NPs, TiO2-NPs, ZnO-NPs

Ligand shell THF Starch or albumine No NoDimension 50-300 nm(aggregates) 5-20 nm 30 nm, 60 nm and 100 nm CuO: b50 nm;TiO2: b100 nm,

ZnO b100 nmBath

Concentration– 5-100 μg/ml 10-1000 μg/ml 10-500 μg/ml

Exposition 75-145 hpf 0-72 hpf 0-96 hpf From stage 8 (5 hpf at 23 °C) tostage 45 (96 hpf at 23 °C)

Specie Zebrafish Zebrafish Zebrafish (dechorionated) XenopusHatching rate – Delay dose-dependent. Two fold

higher instarch-NPs– –

Malformations Arched back, yolksac andheartedema, limitedmovements (dose 5% w/v)

Opaque yolk, benttwisted no to chord,blood accumulation near tail, lower heartrate, pericardialedema, degeneration ofbody part (60% at μg/ml)

Gut malformations and, athigh dose, skeletal muscle fiber separationand jaw defects

ZnO: abnormal gut coil, growing delay;CuO: growing delay, cephalic and organsdefects, gut malformation;TiO2:gut malformations

Mortality Dose dependent High mortality dose-dependent 1 mg/ml 100% Very lowMechanisms

of toxicityTHF decomposition producesγ-butyrolactone, a precursor ofneuro transmitter GABA; probablyitinhibits neuro nalactivity,including breathing

Increase in apoptosis 60 nm more toxic than other NPs,probably because more easily trapped in gutluminalspace; 60 nm dendritic NMs moretoxic than spherical,probably because moresticky; not due to iondissolution

CuO: probably ions dissolution,Cu2+ treatment produce thesame malformations.ZnO: not due to ions dissolution,Zn2+ does not affect embryosdevelopment

Ions dissolution – Ions released butnot responsiblefortoxicity

– –

Localization – Preferentially nuclear; uniform inwhole embryos, with aggregation inskin, heart and brain

Digestive system lume Inside the gut

Reference 80 78 62 49

CuO-NPs,ZnO-NPs, NiO-NPs,Co3O4-NPs

TiO2-NPs PbS-NPs Chitosan-NPs

Ligand shell Alginate No MT or DT NoDimension 200-400 nm Ca 30 nm Ca 30 nm 200 nm or 340 nmConcentration 0.1-200 μg/ml 0.1-5 μg/ml 10-320 μg/ml 5-40 μg/mlExposition 4-120 hpf 2.5-5.5 hpf-120 hpf 5-120 hpf 0-72 hpfSpecies Zebrafish Zebrafish Zebrafish (dechorionated) ZebrafishHatching rate CuO: high decrease(0.5 ppm); ZnO:

high decrease(5 ppm); NiOhigh decrease (50 ppm); Co3O4:no effect

Not affected – Significant decrease(dose dependent)

Malformations No Behavioral changes Both caused bent body axis; PbS-MT also snout,jaw and eye malformation in all surviving embryos

200 nm: dose dependent; developmentdelay, lack of spontaneous movements,yolksac edema, bent trunk, tailmalformation, inflated swimming bladder(5 μg/ml); 340 nm: not statisticallysignificant

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Mortality No No PbS-MT: dose dependent, 100% at 120 μg/ml.PbS-DT no statistically significant mortality

200 nm: dose dependent(100% at 40 μg/ml); 340 nm:dose dependent (15% at 40 μg/ml).

Mechanismsof toxicity

Releasing of freeions; CuO andZnO induce Hsp70transcription

Not due tooxidative stress MT is more instable, leading to ligand desorptionand greater core exposition

Increase in Hsp70 gene expression,apoptosis andreactive oxygen speciesproduction

Ions dissolution Zn2+(8,2 ppm) N Ni2+(5,2 ppm) NCu2+(3,9 ppm) N Co2+(1,2 ppm)after 24 hin embryo culturemedium

– PbS-MT: 4%.PbS-DT: 0.2% –

Localization – – – –Reference 59 77 82 13

Au-NPs TiO2-NPs Au-NPs

Ligand shell MEEE, MEE,MES or TMAT No TMAT or MES or MEEEDimension MES and MEE:0.8 nm and

1.5 nm. TMATand MEEE:0.8 nm,1.5 nm and 15 nm

– MES and TMAT: 1.6 nm;MEEE: 1.3 nm

Concentration MEEE and MES:50 μg/ml.TMAT:0.4 μg/ml

1-1000 μg/ml 0-250 μg/ml

Exposition 0-120 hpf 0-192 hpf 4.5-120 hpfSpecies Zebrafish Zebrafish Zebrafish (dechorionated)Hatching rate – Not affected; TiO2 do not cross

chorion barrier–

Malformations MES: jaw andeye malformations;TMAT: negligible effects;MEE and MEEE: any effects

Opaque yolk, stunting, headedema,no tail

TMAT: 100% (10 μg/ml); MES Ndi MEEE (2 μg/ml)

Mortality TMAT and MES:not significanteffects; MEE and MEEE:anyadverse effects

Dose dependent; in absence of lightno incidence; inpresence of lightsignificant increase of lethality

TMAT: 100% at 100 μg/ml; MES: 40% at 100 μg/mlMEEE:0% at 100 μg/ml

Mechanismsof toxicity

Dose and chargedepending(positivelycharged more toxic)

Phototoxicity (activation of antioxidantgene pathway (Nrf2)); DNA oxidation(increase in 8-OHdG)

Probably TMAT Au-NPs have a higher trappingstrength for metalion transporter and are able to getintocell readily at 24 hpf, perturbing the various transportmechanisms within the cell; trapping strength of MESand MEEE Au-NPs is intermediate and low, respectively.Inflammation, phagocytosis and NO signal transductiongenes are upregulated. Signal transduction of WNT andNOTCH signaling, inflammation IL12, 15, 18, cellcycleG1-S growth, musclecontraction, reproductiongonadotropinregulation, inflammation-histamine signaling, immuneresponse, IL5 signaling gene are down regulated.

Ions dissolution – – –Localization – Diffuse and not specific –Reference 73 79 74

(–): data not available. Abbreviations: BSA: albumin bovine serum; DT: 2,3-dimercaptopropanesulfonate; hpf: hour post-fertilization; MEE: 2-(2-mercaptoethoxy)ethanol; MEEE: 2-(2-(2-mercaptoethoxy)ethoxy)ethanol; MES: 2-mercaptoethanesulfonic acid; MT: 3-mercaptopropanesulfonate; ROS: reactive oxygen species; THF: tertahydrofuran; TMAT: N,N,N-trimethylammoniumethanethiol.

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usually indicate that the toxic effects of NMs are dose-dependent,with high concentrations inducing lethality and lower concentra-tions not affecting viability. In NMVEmodels, it is also possible tomonitor the effects of low NM concentrations, which do not altersurvival, but can nonetheless induce severe malformations orbehavioral impairment.76,77 In a study in Xenopus, althoughtransition metal oxide NPs did not induce mortality at concentra-tions up to 500 mg/L, they did cause significant malformations inthe gut.49 Thus, dose-dependent effects in embryonic survival canbe documented, as reported for Ni-NPs59,62 and for NPs ofbiodegradable materials, e.g., chitosan, which are largely used inmedical applications.13 The most common malformations causedby the uptake of toxicNPs in zebrafish are gastro-intestinal defects,skeletal muscle defects, tail or spinal cord flexure, finfoldabnormalities, yolk sac edema, absence of a tail, twisted notochord,low heart rate, pericardial edema, degeneration of body parts, jawdefects, head skeleton defects, neurological damage and rarelyanencephaly.14,62,78 In Xenopus embryos, growth retardation andcephalic malformation have also been documented.49 Variouseffects in zebrafish development have been reported, such as anincrease in apoptosis, growth retardation most likely due to a delayor inhibition of cell proliferation, impairment of cardiac muscleeither directly or by blockade of the energy sources that influencesmooth cardiac function of embryos.13,78 It has also beendemonstrated that TiO2-NPs induce phototoxicity mediated bythe production of reactive oxygen species (ROS).79 In general,gene expression studies have shown that oxidative stressassociated with an inflammatory response is the basic mechanismby which NPs cause damage in embryonic models.59,69,79,80

Decreased expression of genes involved in cellular differentiation,cell cycle control, reproduction and muscle contraction has alsobeen reported.74

Brain deposition of Ag-NPs can interfere with the control ofcardiac rhythm, respiration and body movements.78 Moreover,the presence of TiO2-NPs in the brain, in low concentrations,causes behavioral changes, such as diminished time spent inswimming with low average velocity and increased burstvelocity of zebrafish embryos. Interestingly, these effects arenot mediated by oxidative brain stress because treatment withantioxidants was not preventive. Rather, there was evidence thatthese abnormalities were most likely due to physical irritationand damage to the gills with defective oxygenation and anoxicbrain damage.77

The importance of NM surface functionalization on nano-particle stability, and thus, on in vivo biological responses maybe efficiently investigated in NMVEs. It has been demonstratedthat, NM interactions in vivo with plasma or other protein-containing fluids, can cause opsonization and agglomeration,which can strongly affect NM side effects.81 A study of zebrafishexamined two different PbS-NPs of nearly an identical core andsize that were functionalized using sodium 3-mercaptopropane-sulfonate (MT) or sodium 2,3-dimercaptopropanesulfonate (DT)ligand. The use of MT ligands was observed to increase themortality and malformation rate in contrast to DT ligands, mostlikely due to the faster decomposition of MT ligands, whichcaused NP aggregation and production of toxic Pb ions.82

NMVE models have also been used to investigate the role ofsurface ion species of NP in nanotoxicity. The toxicity of Cu-

NPs in Xenopus was mainly attributed to dissolved Cu ionsbecause they cause the same toxic effects even at a concentrationthree orders of magnitude less than functionalized Cu-NPs.49 Linet al demonstrated in zebrafish that metal chelators blockedtoxicity mediated by metal NPs, confirming that dissolved ionsplay a role in NP-induced toxicity.59 In contrast to these studies,other reports have shown that Zn49 ions (in Xenopus), and Ni,62

Pb82 and Ag78 ions (in zebrafish) were not harmful or lethal anddid not cause malformations.

The importance of the developing stage

Early stage embryos are more sensitive than more matureembryos.78 When exposure occurs after the larval stage in Xe-nopus or 24 hour post-fertilization (hpf) in zebrafish, NMsaccumulate predominantly in the digestive tract lumen (via oralroute) and only rarely cross the epithelium (transdermalroute).49,62 This is probably due to the presence of tightjunctions in the epidermal barrier, whereas the gut endotheliumis permeable. From the gut, NMs enter the blood stream and arecarried to the liver and heart. Accumulation also occurs in thebrain, probably due to an entry of NMs in embryonic neuro-precursors, rather than passage via the blood-brain barrier.61,78

Using the zebrafish model, it is important to take into account thepresence of the chorionic membrane, which is an acellular three-layered glycoprotein envelope surrounding mature eggs ofteleostean fish.83 The chorionic membrane lasts until larvalhatching at approximately 48-72 hpf. Contradictory results havebeen reported on the passage of NPs through the chorionicmembrane. Some reports have indicated that the chorionmembrane does not retain NPs, which are instead distributedthroughout the entire embryo.14,78,84 In contrast, other reportswere indicative of a protective role of the chorionic membrane inpreventing the uptake, distribution and toxic effects of NPs.79

Other studies have suggested that the chorionic membrane canalso influence the interaction of the embryo with NMs. Forexample, Heiden et al15 reported that embryos exposed to G4dendrimers survived longer if they were dechorionated prior toexposure. A potential explanation for this observation is thepositive charge of the G4 dendrimer, which interacts with thenegative charge of the chorion, resulting in a higher dendrimerconcentration around the embryo and consequently, enhancedNM uptake. A summary of the literature on non-mammalianembryo nano-toxicology is provided in Table 3.

Biomedical application

Imaging and diagnostics

Developmental biological labeling agents can track cells,including their movement and fate during both normal andabnormal embryonic development, as well as provide informa-tion on cellular protein localization, protein-protein interactionsand vascular development. However, classical fluorophores havesome limitations particularly in whole organism studies, due tothe high background noise, photobleaching, low penetrationdepth, low emission and suboptimal spatial resolution.85-87

Similarly, contrast agents such as fluorescent dyes for optical

Figure 3. Use of QDs in embryo microangiography. (A1) QD605, which emits a green color, was used as a contrast agent in the microangiography of thezebrafish embryo; (B1) the Olig2-Dsred transgenic zebrafish line expresses motor neurons and their axon tracts in a red color; (C1) combining the greenfluorescence of QD605 and the red fluorescence of Olig2-Dsred transgenic zebrafish, the vascular network and motor nervous system are displayed together.108

(A2) Whole-embryo fluorescence image after Q705PEGa injection into a CAM vein of a 10-11 day-old chicken; (B2) CAM vasculature and underlying yolkvessels imaged using Q755PEGa; (C2) endocytosed QDs in the endothelial cells of vessels observed on the day after injection. Scale bars in panels A2, B2, andC2 = 25 mm, 1 mm, and 100 μm, respectively.31


coherence tomography88,89 suffer from low resolution and highbackground noise. NMs have the potential to overcome many ofthese restrictions. Current studies are focused on the develop-ment of novel nanoprobes, and NMVEs may represent excellentplatforms to evaluate their performance for in vivo bioimaging.This is exemplified by recent studies of embryonic imagingusing Quantum dots (QDs). QDs are fluorescent semiconductornanocrystals, usually selenides or sulfides of Cd or Zn. Becausethe excitation and emission wavelengths of QDs are exclusivelydependent on crystal size, a broad color gradation can begenerated by tuning the core size and this has been exploited inmulti-labeling experiments.90 In contrast to classical fluoro-phores, QDs provide higher signal intensity and a more enhancedsignal/background ratio. Moreover, they are much more resistantto photobleaching.91 One limitation of QDs in biologicalapplications, which makes their clinical application challengingis their intrinsic toxicity. Concerns are principally raised by thehighly toxic metals that generally constitute the core of QDs,e.g., Cd, Se or Pb, although it is usually coated withbiocompatible molecules.92-94 It has been demonstrated in vivothat QDs are toxic if the NP coating is unstable or degraded,causing core exposure.95,96 Moreover, the toxicity of QDs isdependent upon several features, including size, surface charge,external coating, dose, oxidation and photo-degradation, whichis similar for all NPs.92,97-99 Otherwise, few studies have reportno or slightly toxic effects of QDs in vivo.100-102 NMVE modelshave been usefully exploited in the literature to investigate boththe toxicity and imaging performance of QDs. It has beenreported that encapsulation in copolymers or phospholipidmicelles or even conjugation with streptavidin allows safeinjection and uptake of QDs by embryos with negligible sideeffects.31,100,103 The distribution of unmodified QDs is generallyuniform in all tissues, as demonstrated by studies involvingeither injected104 or exposed embryos.84 In contrast, differentfunctionalization approaches can result in different intracellularlocalizations of QDs. Dubertret et al100 injected QDs encapsu-lated in phospholipid and copolymer micelles into Xenopusembryos (4-cell division), and observed fluorescent accumula-tion in the nuclear compartment after mid-blastula transition,although the mechanism for this localization remains unclear. Insharp contrast, QDs conjugated with streptavidin remained in thecytoplasm both in zebrafish103 and Xenopus embryos.105 QD

functionalization can be tailored to the scope of the experiment.Thus, to study morphogenesis in Xenopus embryos, Stylianouand Skourides105 used QDs to image mesoderm migration invivo. They reported that whereas naked QDs producedhomogeneous staining of the progeny precluding identificationof individual cells, conjugation of QDs to a synthetic biotinylatednuclear localization signal (NLS) peptide allowed the complex toconcentrate in the nuclei, resulting in enhanced contrastimaging.105 Importantly, the authors also reported that thetoxicity of QDs was poorly predictable (i.e., batch-dependent).

A method for labeling cell surface proteins using QDs, basedon protein conjugation to QDs via a polyethylene glycol (PEG)linker, was used by Gussin et al106 to demonstrate in Xenopusoocytes that muscimol (a GABA receptor agonist)-conjugatedQDs could target GABAC receptors expressed on the surfacemembrane of Xenopus oocytes.106 Another elegant method totarget specific proteins by QDs was reported by Charalambous etal, who used QDs to target focal-adhesion complexes in Xenopusembryos. In this experiment, the C-terminus half of the DnaEsplit intein protein was linked to QDs via biotin-streptavidinconjugation in vitro. By injecting this probe and RNA encodingrecombinant Paxillin–EGFP fused to the N-terminus half of theDNAE split intein into blastomeres of 2-cell stage Xenopusembryos, they observed that the QD-based probe was co-localized with Paxillin-EGFP at focal-adhesions.107

QDs were also used as powerful contrast agents for micro-angiography in embryos by virtue of their brightness, which allowsthe visualization of deeper vessels at concentrations 2-3 orders ofmagnitude lower with respect to dextran-FITC (fluorescent dyecommonly used for imaging). The efficacy of QDs as a contrastagent for micro-angiography has been demonstrated in bothzebrafish embryos108 and in the chick chorioallantoic membrane(Figure 3)31. Moreover, QDs accumulate and are retained withinvessel walls, most likely via endocytosis and lysosomal storage.They are thus able to document embryonic vasculogenesis,although their half-life varies with the functionalization processused.31,103 Other distinct advantages of QDs inmicro-angiographyare their increased photostability compared to commonly usedfluorophores and their compatibility with immunohistochemicalprotocols.103

Another imaging application of QDs relates to the measure-ment of cellular and embryonic fluids movements, as

Table 4Summary of NMs and their features applied in non-mammalian embryos imaging.

QDs MNP(Fe3O4) Streptavidin-QDs M-PEG-QDs Ag-NPs QDs or streptavidin-QDs Ag-NPs QDs

Conjugation No No No Muscimol (GABAC

receptor agonist)No No or fibrinogen No No

Size 10-30 nm 20-30 nm – – 5-46 nm – 11.3 nm –Concentration 2.3 μM 1 mg/ml a) 100 nM (1.7 nl)

b) 100 Nm (1.7 nl)c) 1 μM

– 0.19 nM or 0-0,71 nM – 0.2 nM 1 nM

Species Xenopus Xenopus Zebrafish Xenopus Zebrafish Chicken Zebrafish ZebrafishAdministration Injection Feed (24 h) a) injection

b) injectionc) heart injection

Exposure (5 minutes) Exposure (0-2 h or 5 days) Vein injection Exposure (minutes) Injection

Stage 2 cells (1.5 hpfat 23 °C)

35 days a) 0.75 hpfb) 2 hpfc) 32 hpf, 3 days,5 days, 9 days

Oocyte 1.25-2 hpf 10-11 days 21 hpf 0 hpf

Observationtime

Different stageuntil tadpole

36 days a) until 36 hpfb) 36 hpfc) immediately

Oocyte 4 hpf or 120 hpf for dosedependent treatment

11-12 hpf 21 hpf 2 hpf

Toxicity No – No – 0.08 nM no damage;0.19 nM 100% malformation

No – –

Detection Fluorescence MMOCT Fluorescence Fluorescence Dark field SNOMS Fluorescence SNOMS SPIMLocalization All cell types Digestive

tracta) cytoplasm of allembryo cellsb) cell cluster in animal polec) reticular cells of trunkand tail, and vasculature

Membrane homomericGABAC receptor

Retina, brain, heart, gillarches and tail

Vasculature Chorion fluid Entireembryo

Spread No No a) yes during the first cleavageb) noc) yes

No Yes in chorion space Yes Yes Yes

Reference 100 112 103 106 84 31 94 90

NIR-QDs orstreptavidin-NIR-QDs

BaTiO3

nanocrystalAu-NPs QDs CPMV-NPs DEG-Gd2O3-NPs QDs

Conjugation No or NLS No No a) Paxillinb) memGFP

Alexa Fluor 647 No No

Size – 30-90 nm 40 nm – – 17 nm –Concentration – – 5 nM – 50 μg Cells treated

with 3.88 mM1 μM

Species Xenopus Zebrafish Zebrafish Xenopus Chicken Chicken ZebrafishAdministration Injection Injection Injection Injection Injection in CAM Treated cells implant Ventricle injectionStage 32 cells 1 cell 1 cell 2-4 cells 9 days 10 days 32 hpfObservationtime

Stage 10.5(1.5 hpf at 23 °C)

– 6-8 hpf or 22 hpf Stage 8 (5 hpf at 23 °C)or 10 (9 hpf at 23 °C)

16 days 14 days –

Toxicity Some batcheshigh toxic

No No – – – –

10M.Giannaccini

etal

/Nanom


Biology,

andMedicine

xx(2014)

xxx–xxx

Detectio

nFluorescence(N

IR)

Fluorescence

Ram

onmapping

basedon

SERS

Fluorescence

Fluorescence

MRI

Fluorescence

Localization

Mesod

erm

ornu

clei

ofmesod

ermalcells

–Mesod

erm

and

ectoderm

derivativ

esa)

Focal

adhesion

b)mem

brane

–Inducedtumor

Vascularsystem

Spread

No

No

Yes

No

Yes

–Yes

Reference

105

109

111

107

114

113

108

(–):datanotavailable.Abbreviations:C

AM:chorioallantoicmem

brane;CPMV:cow

peamosaicvirus;DEG:diethyleneglycol;G

ABA:g

amma-am

inobutyricacid;hpf:hou

rpo

st-fertilization;MMOCT:m

agneto

motiveop

tical

coherencetomograph

y;MNP;

magnetic

nanoparticle;M-PEG:muscimol-poly(ethylene

glycol);MRI:Magnetic

resonanceim

aging;

NLS:nuclearlocalizationsignal;NIR:near

infra-red;

PEG:

polyethylene

glycol;QDs:quantum

dots;SERS:surface-enhanced

Ram

anscattering;SNOMS:single

nanoparticle

optical

microscopyandspectroscopy

;SPIM

:selectiveplaneillum

inationmicroscopy.


exemplified by the in vivo measurement of the migration ofXenopus mesodermal cells (1.62-2.18 μm/min) as reported byStylianou and Skourides.105 This study demonstrated the freemovement of individual cells inside the cell mass. In a study ofQD movements inside early stage zebrafish embryos, Friedrichet al104 observed faster motion at the 32-cell stage than at the 64-cell stage, when QDs tended to aggregate. This reduced motilitywas most likely due to the closure of the cytoplasmic connectionsbetween giant yolk cells and blastomeres.104

Although QDs offer several advantages compared to classicalfluorescent probes, they still have intrinsic limitations related totheir toxicity. Thus, the use of other substances in the core, suchas copper indium selenide (CIS), is currently being explored toreduce QDs toxicity.94 There are also other limitations related tothe label itself, i.e., dye saturation, bleaching (even if limited),and blinking (fluctuation in fluorescence). A study in zebrafishreported that these limitations could be overcome by secondharmonic generating (SHG) nanoprobes, such as tetragonalBaTiO3.

109 These nanocrystals convert two photons into onewith half wavelength, and thus generate a signal similar to afluorescent dye, but with a narrower emission. They do notbleach or blink and their signal does not saturate. Moreover,similar to QDs, they can be functionalized with antibodies, thusobtaining high detection contrast in immunostaining assays andas a promising tool in biomedical imaging.

Other fluorescent NMs, which have been studied using invivo embryo dynamic imaging, include noble metal NPs, such asAg and Au. At the nanoscale, these NPs have unique opticalproperties, including localized surface plasmon resonance,which is dependent on their size. Moreover, they are morebrilliant than QDs. It has been demonstrated in zebrafishembryos that 9-15 nm Ag-NPs and Au-NPs are passivelytransported into the chorionic space via chorion pore canals upto the inner mass.14,84,110 Generally, the NPs follow simpleBrownian motion, although some remain in chorionic spacecanals for an extended period of time. These trapped NPs serveas nucleation sites and form larger aggregates with incomingNPs.84 Furthermore, the transport of NPs from chorionic spaceinto the embryonic inner mass follows random Brownianmotion, suggesting that the chorion pore channels are poortransport barriers. Au-NPs, once inside the embryo (by diffusionacross the chorionic membrane), are evenly distributed in thederived tissues from all the germ layers.14 These studies haveshown that Au-NPs are able to spread to different blastomeresduring cleavage and subsequently to different organs. However,different results were reported by Wang et al.111 Following aninjection of Au-NPs in zebrafish embryos at the one cell stage,they reported a segregated distribution in the embryonic tissueswith a complete absence of Au-NPs in the yolk tube. It isprobable that the different routes of administration account forthese contradictory results.

The monitoring of Ag-NP and Au-NP movements was usedto measure the viscosity of different regions in the embryos.Regulation of the viscosity gradients of embryonic fluid is one ofthe determining factors of cell differentiation and migration.Using Au-NPs and Ag-NPs of different sizes (and thus color), itwas possible to demonstrate the presence of counter-clockwiseflow patterns and viscosity gradients in the chorionic fluid. The


highest viscosity was observed around the center of the chorionicspace, whereas NPs moved faster near the surface of the chorionlayers and inner mass of the embryos, indicating a lower mediumviscosity.110

Oldenburg et al112 validated the use of magnetic nanoparti-cles (MNPs) as a contrast agent for optical coherencetomography (OCT) in the Xenopus tadpole (a technique referredto as magneto motive OCT, MMOCT). Specifically, they fedtadpoles for 24 hours with MNPs. The application of an externalmodulated magnetic field induced the motion of MNPs, whichmodified the amplitude of the OCT interferogram. Using thismeans, the authors demonstrated that MMOCT considerablyreduced the background signal. As expected, the MNPsaccumulated in embryonic digestive tracts. An improvement inthis imaging method could result from the use of targeted MNPsto label specific structures.

Another application concerns the use of 2-3 nm gadoliniumoxide NPs (Gd-NPs) as a contrast agent for magnetic resonanceimaging (MRI). It has been possible to label glioblastoma cells inxenografts tumors grown in the chicken embryo CAM. The clearand enhanced signal, provided by Gd-NPs, enabled monitoringof cells in vivo during the first hours following implantation andprior to the formation of a tumor or vasculature growth.113 Asummary of the literature on non-mammalian embryo nano-imaging is provided in Table 4.

Delivery of macromolecules

A fundamental step in functional and pathological studies andin therapeutic protein production and gene therapy is theinsertion of exogenous genes into embryos. Because nakedDNA does not enter cells efficiently, transfection experimentsrequire non-toxic gene delivery. Existing methods exploitinggene delivery into embryos include microinjection,115

liposome,116 retroviral and adenoviral agents,117,118 calciumphosphate,119 particle bombardment120 and electroporation.121,122

However, all of these techniques have their limitations: time-consuming, potential oncogenesis, infection risks, immuneresponse, restricted diffusion, limited efficiency, cell structuredamage and embryonic toxicity. Recently, novel methodologiesfor gene delivery based on the use of NMs have been proposed.123-125 Although several NMs have previously been used to transfectcells in vitro, there are only a few studies in the literature reportingon the use of nanocarrier-mediated gene delivery in livingembryos. In contrast to naked fluorescently labeled small DNAmolecules, which diffused rapidly around the cells and were nolonger detected 15 min after injection, Berthault et al126

demonstrated that injection into the intercellular space of differentpolyethyleneimine (PEI)-NPs and cholesterol-NPswas effective indelivering fluorescently labeled small DNAmolecules to 1000-cellstage zebrafish embryos. Specifically, cholesterol-NPs wereretained in the cellular membrane, an observation that could bevery useful for drug or protein membrane targeting. However,these compounds proved to be toxic to embryos, particularly thosecontaining PEI.126 More convincing results were reported by astudy on transgenic chicken production, which injected gelatinNPs as gene carriers intercellularly into the area opaca. Thismethod produced a higher hatching rate compared to virus-

mediated gene transfer, and in addition, provided a higher survivalrate when compared to the use of lipofectamine as a transfectionagent. Thus, gelatin NPs appeared to be promising genenanocarriers, although its current low transfection efficiency andshort gene expression require improvement.8

Vascular remodeling

The control of neo-angiogenesis is a key component forprogress in the treatment of vascular disease and cancer therapy.Stimulation of angiogenesis in adults carries the potential formore effective treatment of ischemic vascular diseases, whileinhibition of neo-angiogenesis is an important therapeuticstrategy in various forms of solid cancers via suppression oftumor growth and the prevention of metastasis. In an interestingreport involving the use of zebrafish embryos to study neo-angiogenesis stimulation, the embryos were injected with amatrigel of poly-lactic acid-glycolic acid (PLGA)-NPs loadedwith 1K1 (an engineered protein derived from the hepatocytegrowth factor/scatter factor HGF/SF), which served as a potentangiogenic agent.127 Significantly higher new vessel formationwas elicited by the 1K1-loaded PLGA-NPs compared to the 1K1alone, although both were accompanied with similar levels ofcell proliferation. This effect may be explained by the sustainedactivation of MAPK signaling via the functionalized nanopar-ticles in contrast to free 1K1.127 A similar result was reportedwith a subcutaneous injection of 1K1-loaded NPs in mice.127

Many embryonic studies have investigated anti-angiogenetictherapy involving NPs. Graphite nanoparticles (multiwalledcarbon nanotubes and fullerenes) have been shown to inhibitVEGF/FGF2-mediated vessel growth in chick embryo CAM.128

However, the mechanisms by which carbon nanoparticles exerttheir anti-angiogenic activity remain unknown, particularly in theexperiments demonstrating the lack of binding and sequesteringof VEGF/FGF2,128 previously reported with Au-NPs.129 Theauthors suggested a potential interaction of the carbon NMs withthe anti-angiogenic factor receptors; however, evidence for thiswas lacking.128 Other studies on the anti-angiogenic effects ofcarbon NMs on cancer therapy reported by Grodzik et al130

concerned the study of carbon NMs in a model of glioblastomamultiform cells cultured on the CAM of the chicken embryo. Inthese experiments, two different carbon-NPs were studied: (1) 2-10 nm NPs produced by the ultra-dispersed detonation diamond(UDD) method and (2) 30-100 nm NPs produced by plasma-assisted chemical vapor deposition using the microwave-radio-frequency (MW-RF) method. Apart from the difference in size,the particles differed in the phase content, as shown by electrondiffraction patterns. Following injection of the particles in theglioblastoma mass, the authors observed a reduction in the vesselarea and in the surrounding chorioallantoic, which correspondedwith decreased tumor growth. The weak differences in theefficacy between the two types of carbon NPs were explained bydifferent inhibitory spectra: carbon UDD-NPs inhibited bothVEGF and FGF-2, whereas carbon MW-RF-NPs significantlyinhibited only VEGF.130 These studies were particularlyinteresting because they exploited intrinsic anti-angiogenicproperties of NPs, which could synergize the therapeutic effectswhen combined with anti-angiogenic molecules.


Another interesting anti-angiogenic approach, which wasexplored in experiments in chicken embryos, involved photo-dynamic therapy (PDT) in choroidal neovascularization (CNV)treatment. PDT therapy consists of the systemic administrationof a photosensitizing drug (PS), which after local light-mediatedactivation, triggers irreversible damage in irradiated tissues. Inclinical use, photosensitizer (PS) drugs have specific drawbacks,e.g., most are hydrophobic and often exert low selectivitytowards pathological neovasculature. This is particularly dan-gerous in the eye, where it is crucial to preserve healthy tissue,during PDT. Vargas et al33 demonstrated that NPs could offernovel solutions. In a model of chicken embryonic CAM, theauthors used PLGA-NPs loaded with a hydrophobic PS andobtained higher vascular occlusion after irradiation, but nodamage to the surrounding healthy tissues compared to thepositive control (PS alone) was observed. Such effects could beexplained by the enhanced distribution of the PS and by theincreased selective retention in diseased vessels, but not inhealthy tissues. This effect was mainly due to the large size of thePS-NPs compared to the PS alone, which was sufficient toprevent leakage from the induced vessel fenestrations. Ex vivoexperiments suggested that PLGA-NPs intrinsically localize invessel walls, thus allowing the PS drug to work moreeffectively.131,132 Although the mechanisms of action are notcompletely understood, the size of the NPs is critical becauselarge NPs are less active than smaller ones because of the slowerPS release and the potential lower uptake by endothelial cells.133

Radioprotection

Radiotherapy used in the treatment of various cancers candamage normal tissues even with adequate protective measuresand optimal treatment protocols. Antioxidant agents have beenused to prevent damage to normal tissues. Amifosine was thefirst approved drug for clinical use.134 Fullerene nanoparticlesalso have potent antioxidant properties due to their free radicalscavenging ability.135 Daroczi et al136 reported that DF1 (watersoluble fullerene derivative) protects zebrafish embryos fromionizing radiation when administered prophylactically similarlyto amifosine. In these irradiated zebrafish embryos, DF1 reducedsignificantly the incidence of midline developmental defects inembryos and restored compromised excretory function. Inaddition, DF1 protected against radiation-induced damage ofzebrafish embryo nerve cells. DF1 acts by significantly loweringROS levels.136 DF1-mediated radioprotection has also beendemonstrated in mice, although to a lesser degree and withoutevidence of selective protection from irradiation of normal asopposed to tumor cells.137

NMVE models in nanomedicine: perspectives andcurrent limitations

The potential effect of nanotechnology on health care isimmense and has ushered in a new era aptly labeled‘Nanomedicine’ in view of the potential benefit in terms ofnovel more effective therapies for various disorders, which arecurrently untreatable. Nanomedicine aims to develop superiormaterials for diagnostic, therapeutic and preventive applica-

tions. Studies on the therapeutic value of a medicinal nano-compound and on nano-toxicity from its use are two-sides of acoin, aimed at the development of more effective therapies toimprove the quality of life. Both in vitro and in vivo studies arerequired in the quest of this objective. In addition, cell culturemodels, although useful, are less informative than studiesperformed in a living organism. The most commonly usedanimal models in medicine are rodents, dogs, pigs and sheep.However, in vivo experiments involving mammals are time-consuming and expensive. Thus, the cost of a complete set oftoxicological assays for a single drug involving hundreds ofanimals is in the range of $1-3 million. As a result, althoughmany engineered nanomaterials look promising for biomedicalapplications, only a few of them have undergone testing inrodents. In addition, the use of mammals for pre-clinical studiesraises several issues and the International Committee for animalprotection and welfare strongly recommends a reduction in thenumber of animals used in experiments.

An alternative solution is to replace the use of mammals withnon-mammalian models whenever possible, or to use non-mammalian testing to prioritize in vivo testing on mammals. Thiswould restrict further testing on mammals only to the mostpromising agents. The results reported in the literature clearlyindicated that NMVEs are as reliable as rodents in the assessmentof NM interactions. The effect of NM core, size, shape,functionalization and ion dissolution in determining the levelof toxicity was comparable to those described in the literatureusing other biological models. As previously reported, NMVEmodels are very sensitive tools to evaluate the interactionsbetween the nanomaterial and a living organism because this canresult in a perturbation of the normal developmental process.These non-mammalian embryos allow easy manipulation andobservation from the earliest stage of development. A largenumber of embryos are produced with each fecundation andthese embryos have a very short early developmental time(zebrafish and Xenopus), thereby incurring much lower costs inhusbandry/housing. Such tests would require small quantities oftest material, which could be crucial to foster the use of NMVEsin medium- and high-throughput systems for fast, cheap andaccurate nanosafety testing. Using these systems, studies wereable to assess toxicity, teratogenic effects, identify the majormechanisms of toxicity and perform hazard ranking. However,achieving this goal requires further development. From theethical perspective, the exposure time to NMs should rangefrom fecundation to the time at which they become capableof independent feeding. Many published reports haveaddressed this timeframe,13,49,59,62,73,74,77,78,82 but there areonly a few studies in which this time was prolonged by severalhours to ensure sufficient NM uptake by the digestive tract.79,80

In some reports, enhanced NM uptake was achieved by embryodechorionation.62,73 Defined standards that incorporate the bestNM exposure time, dechorionation, sacrifice time-point andnanosafety risk assessment methodologies are urgently needed.Concerning the assessment of toxicity after NM exposure inembryos, the survival and malformation,13,49,59,62,73,74,77-80,82

abnormal behavior,13,77 gene expression perturbation,13,74 oxi-dative stress13,79 and cell death mechanisms13,78 were mainlyexamined.


Recent advances in genome sequencing have demonstratedthe strong conservation of genes across the animal kingdom, andthe similarity between human and non-mammalian genomes.Non-mammal vertebrates are not only convenient models butalso share physiological and pharmacological properties withhumans, including several genes and molecular pathways. Inparticular, the zebrafish model has a great homology with thehuman genome (72% of protein encoding genes, 82% of whichare genes associated to human diseases138). The zebrafish offersthe distinct advantage that libraries of mutagenized individualscan be created. For this purpose, a dedicated Zebrafish MutationProject was initiated, aimed at creating a knockout allele in everyprotein-coding gene in the zebrafish genome. Using a combina-tion of whole exome enrichment and Illumina Next GenerationSequencing,139 mutant alleles generated were characterized,archived and could be requested from the Zebrafish InternationalResource Center (ZIRC). Thus far, approximately 11892 geneshave been mutated (45%), and the project is expected to beconcluded in a few years.139 In the near future, the researchcommunity could have access to a large library of thousands ofmutants enabling testing of specific nanodrugs on moreappropriate models, with respect to safety, pharmacokinetics,biodistribution and efficacy. Currently, the main limitation ofthis system is the lack of resources (e.g., species-specificantibodies), but this trend could change rapidly because of thegrowing interest of the scientific community.

X. laevis offers alternative and complementary features forexperimental studies. Despite the difficulty in the production oftransgenic animals, their larger size allows easy manipulation,microinjection and microsurgery. The first division splits theembryo's right side from the left side, and after the followingdivision, the dorsal and ventral blastomeres are clearlydistinguishable.140 This strictly regulated pattern of cell divisionin Xenopus is very useful because it is possible, during NMsmicroinjections, to employ the contralateral non-injected sideas an internal control. In particular, some organs are distinctlyderived from dorsal or ventral blastomeres, providing a meansto investigate the fate of the injected NPs. Moreover, Xenopusis a powerful tool for multipotent cell assays performed intransplant experiments. Xenopus “animal cap” cells are multi-potent cells situated at the core of the animal pole of theblastula or at the very early gastrula-stage embryo.141 Animalcap cells can be isolated from an embryo and cultured in vitrowith the NMs, prior to transplantation into an untreatedembryo for further assays (M. Giannaccini, personalcommunication).

Finally, the chorioallantoic membranes obtained fromdeveloping chicken eggs represent an excellent model to testthe safety or efficacy of various nano-formulations forapplications related to angiogenesis and tumor growth.

In conclusion, NMVE holds the potential to become verypopular alternative organisms and may be extensively usedmodels in nanomedicine. Multiple applications in the field ofbioimaging, delivery of macromolecules, neo- and anti-angio-genic activity and radioprotection have been previouslyproposed. We propose that NMVE model systems will offeropportunities for the progress of nanotechnology productsin medicine.

Acknowledgments

The authors would like to thank Irene Giannaccini andAndrea Berni for the line drawings.

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Non-mammalian vertebrate embryos as models in nanomedicine

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